Interaction of melanoma and breast cancer brain seeding TCs with VWF. (A) Experimental design of in vivo MPLSM imaging in anti-VWF–treated mice vs isotype control antibody–treated mice. Treatment was started 30 minutes before TC injection, then given once per day, and then following the imaging schedule after day 3. Late antibody treatment started on day 10. Individual arrested TCs were followed throughout the brain metastatic cascade until day 28 after TC injection. (B) Representative images of intravital VWF immunofluorescence staining with anti-VWF antibody on day 3 after TC injection. It was obligatory to evaluate a clear green fluorescence signal accumulation (arrowhead) as positive VWF activation (VWF relative fluorescence intensity ∼1.5×; semi-quantitative analysis). Faint background vessel signal represents unbound FITC-labeled antibody, creating an angiogram. Scale bars: 10 µm. (C) Mouse brain slides. Representative images with immunofluorescent staining of an arrested intravascular A2058 melanoma cell (arrow) and possibly extravasating TCs (arrow heads) and their interaction with VWF fibers (green). 4,6 Diamidino-2-phenylindole (DAPI) staining in blue. Scale bars: 50 µm. (D) Human brain slides. Images with immunofluorescent staining of human perimetastatic brain tissue around a breast cancer brain metastasis. Abundant VWF fibers (green) are detectable in associated vessels and endothelial cells. DAPI staining (blue) and platelet staining (red). Scale bars: 50 µm.